Towards the control of biofilm formation in Anabaena (Nostoc) sp. PCC7120: novel insights into the genes involved and their regulation

Abstract

Cyanobacteria are major components of biofilms in light-exposed environments, contributing to nutrient cycling, nitrogen fixation and global biogeochemical processes. Although nitrogen-fixing cyanobacteria have been successfully used in biofertilization, the regulatory mechanisms underlying biofilm formation remain poorly understood. In this work, we have identified 183 novel genes in Anabaena sp. PCC7120 potentially associated with exopolysaccharide (EPS) biosynthesis and biofilm formation, unveiling conserved and novel regulatory connections shared with phylogenetically distant bacteria. Anabaena possesses homologues of two-component systems such as XssRS and ColRS from Xanthomonas spp., and AnCrpAB from Methylobacillus, suggesting that these homologues play essential or advantageous roles in biofilm formation across diverse bacterial lineages. Additionally, Anabaena features homologues of several proteins exhibiting the GG-secretion motif typical of small proteins required for biofilm formation in unicellular cyanobacteria. A wide array of biofilm-related genes in Anabaena, including major gene clusters participating in the synthesis and translocation of EPS and key regulatory proteins involved in the control of biofilms in other bacteria are modulated by ferric uptake regulator proteins. These findings link the control of biofilm formation in Anabaena to environmental cues such as metal availability, desiccation and nitrogen levels, providing new insights to improve the use of nitrogen-fixing cyanobacterial biofilms in sustainable agriculture and environmental management.

Keywords: GG‐secretion motif; biofilms; cyanobacteria; exopolysaccharides; ferric uptake regulator; two‐component systems.

Fig. 1

Identification of potential Anabaena sp. PCC7120 homologues to Synechococcus elongatus PCC 7942 EbfG1‐4. (a) GG‐motif alignment of EbfG1‐4 and Anabaena sp. PCC7120 GG‐motif‐containing proteins previously identified by Wang et al. (2011). (b) Full sequence identity matrix of the potential homologues. The best matches for each EbfG protein are outlined in blue. (c) Anabaena sp. PCC7120 clusters most likely to correspond to the ebfG1‐4 cluster based on identity and published data, taking into account cds potentially involved in their processing, modification and secretion. Groups of GG‐motif‐containing proteins displaying a higher identity and genomic proximity to each other are highlighted in the same colour in (a, b). Raw data for (b) can be found in Supporting Information Table S5.

Fig. 2

Predicted amyloidogenic hotspots in the protein sequences of potential Anabaena sp. PCC7120 homologues to Synechococcus elongatus PCC 7942 EbfG1‐4, represented as darker sections. Hotspots were determined based on prediction consensus of at least three of five bioinformatic methods applied (APPNN, Waltz, Aggrescan, ArchCandy and TANGO). Genes belonging to one gene cluster are represented in the same colour. A more detailed representation of each method's results is available in Supporting Information Fig. S1.

Fig. 3

Distribution of putative ferric uptake regulator (FUR)‐binding boxes throughout the eight gene clusters involved in exopolysaccharide synthesis in Anabaena sp. PCC7120 described by Potnis et al. (2021). Red, blue and green represent Fur or iron uptake regulator (A), zinc uptake regulator (Z) and peroxide response regulator (P) regulation, respectively. Coloured backgrounds indicate the presence of a binding box directly upstream of a predicted operon according to the MicrobesOnline operon tool, indicated by a black arrow spanning the putative transcriptional unit.

Fig. 4

In vitro interaction between Fur or iron uptake regulator (FurA) and the promoter regions of a selection of genes, evaluated through electrophoretic mobility shift assays. All assays were performed with DNA fragments free or incubated along with the indicated increasing concentrations of FurA (nM). An internal fragment of gene pkn22 (ifpkn22) was used as nonspecific competitor DNA.

Fig. 5

In vitro interaction between zinc uptake regulator (Zur) and the promoter regions of a selection of genes, evaluated through electrophoretic mobility shift assays. All assays were performed with DNA fragments free or incubated along with the indicated increasing concentrations of Zur (nM). An internal fragment of gene pkn22 (ifpkn22) was used as nonspecific competitor DNA.

Fig. 6

In vitro interaction between peroxide response regulator (PerR) and the promoter regions of a selection of genes, evaluated through electrophoretic mobility shift assays. All assays were performed with DNA fragments free or incubated along with the indicated increasing concentrations of PerR (nM). An internal fragment of gene pkn22 (ifpkn22) was used as nonspecific competitor DNA.

Fig. 7

Impact on biofilm formation of deregulation of the Anabaena sp. PCC7120 ferric uptake regulator (FUR) paralogs. Strains used include the wild‐type (WT) Anabaena and variant strains AG2770FurA (FurA‐OE), EB2770FurC (PerR‐OE), Δzur or VCS2770FurB (Zur‐OE). OE, overexpressing. (a) Representative crystal violet‐stained biofilms grown on Ibidi uncoated eight‐well plates. For every strain, each well corresponds to an individual experiment. (b) Image quantification of stained biofilm per well, with integrated density as proxy for biofilm biomass. Values correspond to five independent experiments consisting in two individual wells each, normalized to the average integrated density of the WT strain. Median and quartiles are represented with dashed and dotted lines, respectively. The mean value is included under each dataset. Inset: magnified view of the PerR‐OE and Δzur data. Statistical significance determined through a Kruskal–Wallis test is represented as follows: ns, non significative, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.